Title: To investigate the effectiveness of antibiotics in inhibiting growth of bacterial cell.

Antibiotics do not work on viruses because viruses are not alive. A bacterium is a

living, reproducing life form. A virus is just a piece of DNA (or RNA). A virus injects its

DNA into a living cell and has that cell reproduce more of the viral DNA. With a virus there

is nothing to "kill," so antibiotics don't work on it.

Problem statement

Which plant has the highest antimicrobial properties?

Objective

To study the effectiveness of different antibiotics on inhibiting bacteria (Staphylococcus and

Escherichia Coli) growth.

Aim

To investigate the antimicrobial properties of plants.

Hypothesis

Garlic has the highest antimicrobial properties among ginger, mint, cinnamon, cumin, and

turmeric plant extracts.

Apparatus Materials

1. Paper towel 1. Sterile nutrient agar

2.1 Bunsen burner 2. 1 Ginger piece

3.1 Bench spray of disinfectant 3.1 garlic clove

4.2 Sterile Petri dish with cover 4.Cinnamon extract

5. 1Marker pen 5. 1 Turmeric piece

6. 1sterile forceps 6. 1 Mint leave

7. 6 Paper disc 7. Cumin extract

8. 1 Pipette 8. Staphylococcus bacterium

9. 1 pestle and mortar 9. 1 Hand wash (Dettol solution )

10. Incubator set at 30°C 10. Distilled water

11.1 petri dish without cover 11. Alcohol

solution

Procedure:

A. Variables and techniques

Variables How variables are conducted(techniques)

Manipulated: Different types of antibiotics such as Ampicilin, Carbenicilin,

Type of antibiotic used on Streptomycin, and Tetracycline were used

staphylococcus
Responding: Diameter of clear zone produced after 24 hours were measure with a

Diameter of clear zone ruler. Diameter is proportional to the effectiveness of antibiotics on

the bacteria.
Constant:

Temperature of incubation, The culture was incubated in the same temperature of 30°C.

Amount of antibiotics, A pipette was used to measure the same amount of antibiotics and

Type and size of agar plate placed on the Petri dish on the solid agar.

used, The same size of agar plate was used in the experiment
time kept in incubator Bacteria with antibiotics were kept in the incubator for the same

amount of time-24 hours.

B. Conducting experiment

1. Hands were first sterile by washing with the Dettol solution and the working area was

thoroughly sprayed with the disinfectant. Sprayed area was left for 10 minutes and

then wiped with paper towels. This can prevent any microorganism residing to affect

the experiment condition as the harmless Staphylococcus can be transformed to

pneumonia causing cells if they are in contact in medium containing dead broken

open cells of pathogenic strain.

2. A Petri dish was labeled with alphabets G, C, T, and W representing ginger, cumin,

turmeric and distilled water respectively using a marker pen. Figure 2 shows how the

Petri dish was labeled. The spacing between the labels must be approximately equal to

prevent any overlapping of the plant extract.

3. Plant extract was obtained by crushing plant product in a mortar with a pestle. Before

this is done, the pestle and mortar is first rinsed with alcohol. The purpose using

alcohol instead of water is because alcohol is likely to kill any bacteria that might

otherwise contaminate the extract.

4. Individual discs were used to soak the garlic, ginger, cinnamon, cumin, turmeric and

mint separately and they are later place on the Petri dish without cover. This is to

ensure that the dish does not contain excess water from the plant extract.
5. By using a pipette, 200μl of inoculums containing the Staphylococcus were

introduced into the Petri dish.

6. Once agar is liquefied by placing the bottle in hot water bath, it is removed and

allowed to cool to about 50°C, a temperature where we can handle the bottle easily

and also to reduce the amount of condensation when agar is poured on the Petri dish.

7. The mouth of a conical flask filled with agar was heated up approximately to 45°C.

This is to kill the entire microorganism present in the mouth of conical flask from

contaminating the Petri dish.

8. The Petri dish was platted by pouring the sterile nutrient agar carefully into the dish

covering about ¾ volume of the dish. The lid was immediately closed and the Petri

dish was swirled in a motion of 8 to ensure a fast cooling and to prevent the agar from

touching the other part of the Petri dish. It was leaved for few minutes for extra

surface liquid to be absorbed.

9. The forceps were flamed to sterilize it and used to pick up a disc soaked with water.

The lid of Petri dish was raised and the disc was placed firmly in the centre of agar

with labeled W at the base. The lid was raised as minimum as possible to prevent

much air entering the agar solution as air surrounding might contain microorganism

that can affect the results of the experiment.

10. The dry individual disk which had been soaked with the ginger, cumin, turmeric and

mint were placed on the agar and spaced evenly parallel with the labeled alphabets.
11. Steps 2 to 9 were repeated with 3 different plant extract that is garlic, cinnamon and

on Staphylococcus.

12. The Petri dish was closed and the entire Petri dish was turned upside down. This

practice prevents condensation that collects on the lid from contaminating the bacteria

on the agar. The Petri dish was not taped all around because this might lead to

growth of anaerobic bacteria some which may be harmful and also exclude oxygen

needed for bacterial growth in the agar.

13. The Petri dish is placed in the incubator keeping it in the inverted position at a

constant temperature of 30°C.

14. Hands were washed with Dettol solution and the working area was cleaned again with

the disinfectant spray.

15. 24 hours after incubation, the plate was observed without opening them. The area

where Staphylococcus has grown will remain opaque but the place where bacteria

have inhibited growth will become clear. This is the inhibition zones.

16. The diameter of the inhibition zones were measured in millimeters using a ruler and

recorded.

17. All Staphylococcus bacteria were destroyed by using an autoclave on all

contaminated items.
18. The data’s obtained were tabulated. Data from other members of the class who use

turmeric, cumin, ginger, cinnamon, garlic and mint on Escherichia coli was collected

and compared. A graph was drawn comparing the effectiveness of different plant

extract on Staphylococcus.

G C

Figure 1 : The top view of the labeled Petri dish

Observations and results

A. Tabulation of data

Types of antibiotics
Ampicilin Carbenicilin Streptomycin Tetracycline water
Reading 1(mm) 28.0 38.0 18.0 27.0 0.00
Reading 2(mm) 30.0 38.0 20.0 27.0 0.00
Reading 3(mm) 30.0 40.0 19.0 27.0 0.00
Average reading (mm) 29.3 38.7 19.0 27.0 0.00
Table 1: The diameter of the inhibition zones using Staphylococcus

Types of antibiotics
Ampicilin Carbenicilin Streptomycin Tetracycline water
Reading 1(mm) 30.0 21.0 15.0 24.0 0.00
Reading 2(mm) 31.0 21.0 19.0 25.0 0.00
Reading 3(mm) 33.0 22.0 20.0 23.0 0.00
Average reading (mm) 31.3 21.3 18.0 24.0 0.00
Table 2: The diameter of the inhibition zones using Escherichia Coli

B. Graph of diameter of inhibition zone using Escherichia Coli and

Staphylococcus
 45
40
35
30
25
20 Diameter of inhibition zone
using Staphylococcus
15
Diameter of inhibition zone
10 using Escherichia Coli
5
0
li n
il i
n in in er
ci ic ys y cl at
pi n om ac w
m r be pt tr
A
Ca tr e Te
S

Discussion

A. Explanation of results

Which antibiotic is most effective on Staphylococcus?

The most effective antibiotic on Staphylococcus was Carbenicilini because it has the

largest diameter of clear zone which is 38.7mm. The diameter of clear zone is proportional to

the effectiveness of antibiotics. Streptomycin was the least effective antibiotics because it

produces the smallest diameter of clear zone which is 19.0mm. Tetracycline and Ampicilin

produces diameter of clear zone 27.0mm and 29.3mm respectively. The antibiotics are

increasing effective with Carbenicilin ˃ Ampicilin˃ Tetracycline ˃ Streptomycin.

Which is antibiotic is the most effective and least effective on both Staphylococcus and

Escherichia Coli. ?
 Ampicillin is the most effective antibiotics on both the bacteria Staphylococcus and

Escherichia coli. This is because, Ampicillin is the most effective antibiotic on Escherichia

Coli and the second most effective on Staphylococcus. The diameter produced by Ampicillin

on Escherichia Coli was the biggest, thus indicating the effectiveness of the antibiotics. The

diameter produced by Ampicillin on Staphylococcus was the second largest.

The least effective antibiotic on both Escherichia Coli and Staphylococcus is

Streptomycin as the diameter of inhibition zone produced on the agar was the smallest.

Both bacteria were more resistant with Streptomycin.

The area around the distilled water disc was not affected. There were no clear zones

produced in both experiments. So, distilled water act as a control.

Bacteria show resistance to distilled water.

Staphylococcus is not resistant to Carbenicilin. Thus, the

peptidoglycan wall is broken easily and the rate of reproduction of

Figure 3: Structure of Carbenicillin

Staphylococcus was stopped by the antibiotic Carbenicilin. These

variations of sensibility are due to characteristics of indicators strains

(presence or absence of receiving sites or immunoproteinii). Carbenicillin exerts its

antibacterial activity by interference with final cell wall synthesis of susceptible bacteria.

This inactivation of the enzyme prevents the formation of a cross-link of two linear

peptidoglycan strands, inhibiting the third and last stage of bacterial cell wall synthesis. Cell

lysis is then mediated by bacterial cell wall autolytic enzymes such as autolysins.

Tetracyclineiii are protein synthesis inhibitors that interrupt or prevent the transcription

and translation of microbial genes causing the genetic codes to be misread and the wrong

protein made in the bacterial cell.

 Streptomycin is bactericidal in action. It inhibits protein synthesis by combining

irreversibly with the 30S subunit of the70S ribosome, found typically in prokaryotes.

Specifically, it binds with the S12 protein involved in the initiation of protein synthesis.

Streptomyciniv is also known to prevent the normal dissociation of 70S ribosome into their

50S and 30S subunits. Thus formation of polysomes is inhibited. The initiation of protein

synthesis by blocking the binding of initiator N-formylmethionine tRNA to the ribosome is

also prevented. The overall effect of streptomycin seems to involve distorting the ribosome so

that transition from initiation complex (30S-mRNA-tRNA) to chain elongating ribosome is

blocked. Thus, the normal sequence of translation is disrupted, the bacteria is unable to

synthesize proteins vital for its cell growth and thereby fails to survive. The drug also disrupts

the cell membrane of susceptible bacteria.

A compromise temperature of 30°C was used because this is the optimum

temperature for bacteria growth. A high temperature beyond 37°C will increase reproduction

rate of bacteria. Therefore, clear zones cannot be measured accurately. A low temperature

will suppress enzymes in bacteria and decreases the reproduction rate of bacteria. Thus, we

will need to wait longer to see the results.

Only four antibiotics were used in one Petri dish to prevent overlapping of the

inhibition zones. Bacteria used were Escherichia Coli and Staphylococcusv. This is because

they are non-pathogenic, can be obtained easily and can be cultured easily.

Nutrient agar was used as a medium of growth of the Staphylococcus species because

most microorganisms need a good source of carbon, nitrogen and mineral resources.

Only one hand is used in handling the apparatus to prevent the transmission of

microorganism or the antibiotics to the culture medium. Washing hands before experiment
will minimize contamination of the bacteria cultures. Washing hands after experiment will

minimize exposure to harmful bacteria that may be growing in the cultures.

B. Conclusion

Carbenicilin is the most effective antibiotic on Staphylococcus bacterium because it has the

largest diameter of clear zone. Ampicillin is the most effective antibiotics on both the bacteria

Staphylococcus and Escherichia coli. The least effective antibiotic on both Escherichia Coli

and Staphylococcus is Streptomycin. The diameter of clear zone is proportional to the

effectiveness of antibiotics.thus, the hypothesis was accepted.

Limitations

The genetic makeup of the bacteria cells used might not be exactly the same. The

resistances of the species of bacteria used will therefore not the same. Both bacteria were

more resistant with Streptomycin. Gene in bacteria alters the intracellular target protein for an

antibiotic molecule, reducing its inhibitory effect. So, the bacteria have resistance gene on

antibiotics Ampicilin, Carbenicilin, Streptomycin, and Tetracycline. Exposing bacterial

population to specific antibiotics will kill the antibiotic sensitive bacteria. However

sometimes, mutation may arise that leads the bacteria to have more resistance on the

antibiotics. This factor can affect the results of the experiment.

 In addition, there is humidity within the Petri dishes. Slight change in humidity can

affect the rate of reproduction of bacteria. When the bacterium was inoculated into the Petri

dish some air will eventually escape into the dish. Moreover, the bacteria might not be

distributed evenly throughout the dish.

Further work

1. Investigate the effectiveness of the four types of antibiotics on protozoan cells like the

Amoeba cells.

2. Investigate the influence of very high temperature on antibiotics.

3. Investigate the effectiveness of antibiotics on viral particles

(2817 words)
i
Carbenicillin: Chemistry and Mode of Action,Kenneth Butler, Arthur R. English, Verne A. Ray and A. E.
Timreck,The Journal of Infectious Diseases, Vol. 122, Supplement. Symposium on Carbenicillin: A Clinical
Profile (Sep., 1970), pp. S1-S8 ,(article consists of 8 pages) ,Published by: The University of Chicago Press,
Stable URL: http://www.jstor.org/stable/30108303

ii
http://www.pjbs.org/pjnonline/fin202.pdf

S. B. Vakulenko and S. Mobashery, Versatility of Aminoglycosides and Prospects for Their Future
iii

Clin. Microbiol. Rev., July 1, 2003; 16(3): 430 - 450.

Streptomycin –: A Highly Potent Antibiotic,

iv

http://microbiology.suite101.com/article.cfm/sterptomycin_#ixzz0vyz1FN66

v
http://www.merck.com/mmhe/sec17/ch192/ch192a.html